Sensing system for specific substance and molecule detection

ABSTRACT

A system for detecting a specific substance or analyte of interest is provided that includes one or more sensing units and an instrument for analyzing the sensing units. Each sensing unit preferably includes a substrate, an attachment layer and at least one capture layer that includes a ligand layer. In one embodiment, the attachment layer is tripartite and includes a lower binding surface held to the substrate and an upper binding surface that holds the ligand layer, together with an insulating layer disposed between these two surfaces. The lower binding surface provides a durable and stable attachment to the substrate. The upper binding surface holds the ligand layer and does not jeopardize the integrity or viability thereof. The insulating layer prevents any unwanted interaction between the lower and upper binding surfaces. Each sensing unit is supported on a test piece received by the instrument. The instrument controllably positions the test piece using marks and/or codes on the test piece. The instrument measures a difference in mass in the sensing unit in connection with determining whether or not the analyte of interest is present. A light beam is used in making this measurement. In one embodiment, multiple reflections of the light beam on the same sensing unit are utilized to improve the sensitivity of the instrument. Mass enhancement techniques are also preferably utilized to effectively amplify the detected signal that is indicative of the analyte&#39;s presence. The system can also include a device for heating, humidifying and mixing materials of the sensing units, as well as preventing cross-contamination of the sensing units.

FIELD OF THE INVENTION

[0001] The present invention relates to detection and/or quantitation ofan analyte of interest or specific substance and, in particular, to asystem that includes an instrument for measuring mass associated with amulti-layered sensing unit to determine whether an analyte of interestis present.

BACKGROUND OF THE INVENTION

[0002] Sensors and methods for the detection of the presence ofsubstances or molecules in a sample, using a solid-phase assay system,have been described previously. Typically, the sample is put in contactwith the sensor, allowing analyte present in the sample to bind to theanalyte-specific ligand layer of the sensor. For analysis, the samplemay be removed from the sensor. The sensor surface is then analyzed forthe presence of the analyte. Sensors can be defined as includingimmunosensors, affinity sensors and ligand binding sensors, each ofwhich is characterized as involving specific mass change activity inconnection with determining whether or not certain molecules orsubstances are present. Sensors are typically efficient at binding thesubstance of interest (analyte) and highly sensitive and specific to theanalyte. The sensor may consist of one or several layers of variouschemical and physical compositions. The composition depends on thenature of the analyte and the matrix in which the analyte is contained.These layers may include any combination of: a solid supportingsubstrate; attachment layer or layers to bind the substrate and/orsubsequent layers in the sensors any number of intermediate layers; aligand layer that binds specifically to the analyte. Detection of theanalyte bound to the sensor can be achieved by several means including,but not limited to, electrochemical, chemical, and optical methods.Detection of the analyte may be enhanced by various means includingenzyme amplification, and the use of a mass-enhanced analyte-specificsecondary ligand.

[0003] A solid substrate, or base, of the sensor has inherent physical,chemical, electrical, or optical properties that are suited specificallyto the detection method employed in the assay. A ligand layer istypically provided above the substrate and an analyte to be detected ormeasured is bound to the ligand layer. The solid support may be used forthe direct binding of the analyte to its surface and subsequentdetection. However, depending on the composition, complexity, and/orstability of the analyte or the sample in which the analyte iscontained, and the nature of the interactions of the sample/analyte withthe solid substrate, it may be necessary to add one or severalintermediate layers to the solid support.

[0004] Attachment layers may be used as intermediate layers between thesolid substrate and the ligand layer if, for example, the ligand layerdoes not adhere to the substrate, or is destroyed, denatured,destabilized or otherwise inactivated upon binding to the substrate. Thesurface of the intermediate layer in contact with the solid substratemust adhere tightly to the substrate throughout the preparation and useof the test piece. The surface of the intermediate layer opposite thesolid substrate must either be suitable for strong binding of either theligand layer or another intermediate layer. In this manner, and usingthese considerations for the nature of the intermediate layers, multiplelayers may be assembled, the topmost of which is suitable for thebinding of the ligand layer.

[0005] The ligand layer forms a sensing surface that is receptivespecifically to the analyte of interest when the analyte is present in asample to be tested. The analyte is thus immobilized onto the sensingsurface of the sensor and can be detected by any of the methodsmentioned above.

[0006] Although some multi-layered sensors such as those outlined abovehave been described previously in the prior art, development of suchsensors, in accordance with the present invention, seeks to improve andenhance their sensitivity. The goal of one element of the presentinvention, in order to improve this sensitivity, is to immobilize aligand layer that retains maximum binding capacity for a specificanalyte. This usually involves the use of an intermediate attachmentlayer or layers as described above. This multi-layer molecular film isdesigned specifically to accommodate downward interaction with thesubstrate, and upward, optimized interaction with the ligand layer.

[0007] Detachment, or delamination, of these intermediate layers fromthe supporting substrate is a serious problem that must be solved tosuccessfully build a multi-layer sensing surface. Delamination occursbetween the substrate and an intermediate layer if the composition ofthese two components is not conducive to a strong physical or chemicalinteraction between the components. The interaction between thesubstrate and intermediate layer may be weakened during the manufacture,assembly, transport, preparation or use of the sensor.

[0008] Once an attachment layer or layers is produced that is stable todelamination from the solid support, the topmost of the intermediatelayers is used to immobilize a ligand layer specific to the analyte ofinterest. It is critical that the topmost attachment layer optimizes itsinteraction with the ligand layer in order to provide maximum bindingcapacity for the analyte of interest and prevent denaturation,deactivation, or inactivation of the ligand layer. These provisions forthe immobilization of the ligand layer are essential to the enhancedsensitivity of the test.

[0009] Known sensing systems, in addition to multi-layered sensors forimmobilizing analyte that may be present in a test sample, includeinstrumentation for detecting the immobilized analyte. One class ofinstrumentation, including surface acoustic wave spectroscopy,ellipsometry, and quartz microbalances, measures the change in mass ofthe sensor upon immobilization of an analyte. Generally speaking, whenthe analyte is present in the test sample, it can be detected based on achange in mass at the surface as compared to the mass when no analyte ispresent in the sample.

[0010] Optical instruments in this class, as described in the prior art,direct a beam of light through a number of instrument components orelements to the sensing surface that has been previously exposed to thesample being tested. Light is reflected from the sensor, and itsreflected properties, including intensity and various optical propertiesmay be measured. Any change in mass of the sensor due to analyte bindingis represented by a change in the properties of the reflected light. Inparticular, measuring changes in polarization state of the reflectedlight has proven to be a highly sensitive measurement of mass change.Briefly, the sensor may be analyzed by an appropriate instrument thatdetects and/or measures the presence of the analyte using lightreflected from the analyte or light that is transmitted through theanalyte.

[0011] The main problem associated with instruments that employ theseoptical techniques for detection of surface bound analyte involves theaccuracy of detection. Since extremely small changes in mass may beindicative of the presence of the analyte, it is a goal of the presentinvention that the instrument be highly sensitive to enable it to detectsuch mass changes. Additionally, because these instruments are expectedto be utilized in a variety of environments outside of well-controlledlaboratory settings, it is a further goal that the instrument designtake into consideration a number of factors such as component size,durability and automation of instrument operation.

[0012] Based on the foregoing factors and considerations, it would beadvantageous to devise a sensor system that overcomes such drawbacks ordeficiencies of the prior art by providing a system that includes aninstrument that readily functions and cooperates with a sensing unit fordetection and/or measurement of specific mass change activity due to thepresence of an analyte of interest. The instrument of such a systemwould be highly sensitive and accurate in connection with the detectionand/or measurement related to mass change, while the sensing unit ofsuch a system would immobilize a ligand layer that retains, for allnecessary purposes, the analyte of interest.

SUMMARY OF THE INVENTION

[0013] In accordance with the present invention, a sensing system isprovided that includes a sensing unit and associated processes formaking, assembling, and using such a sensing unit. The sensing systemalso includes a test piece on which is positioned one or more sensingunits capable of capturing and immobilizing an analyte or analytes ofinterest from a test sample, and an instrument for detecting theanalyte(s) immobilized on the sensing unit. The test sample containingthe analyte of interest may take the form of a true vapor, a liquid oran extracted solid.

[0014] In one embodiment, the sensing unit includes a solid reflectingsubstrate, an analyte-specific ligand layer and a tripartite attachmentlayer used to immobilize the ligand layer. The solid substrate ispreferably any reflective substance other than a free electron metalthat provides a base of the sensing unit. The upper surface of the solidsubstrate would, if improperly isolated from the ligand layer, causedenaturation, decay, or inactivation of the ligand layer. The tripartiteattachment layer is defined by a lower binding surface, an insulatingintermediate layer, and an upper binding surface.

[0015] The tripartite attachment layer used to immobilize the ligandlayer may consist of three distinct materials, or different materialcompositions of a single material, providing that each element of thislayer (the lower binding surface, the upper binding surface, and theinsulating layer) has properties that are suited particularly for itsrole in the tripartite attachment layer.

[0016] The lower binding surface contacts the surface of the solidsubstrate and is comprised of a material that adheres tightly to thesolid substrate. This lower binding surface provides a durable andstable lamination of the entire attachment layer to the silicon surface.The upper binding surface of the tripartite attachment layer haschemical or physical properties that allow it to immobilize ananalyte-specific ligand layer. The insulating layer is located betweenthe lower and upper binding surfaces. Significantly, the insulatinglayer acts to prevent the transfer of various effects from the lowerbinding surface to the upper binding surface and, additionally, from theupper binding surface to the lower binding surface. The insulatingintermediate layer protects the ligand layer from the various effects ofthe solid substrate. The analyte-specific ligand layer is in contactwith the upper binding surface of the tripartite attachment layer. Thisligand layer captures and immobilizes a specific analyte when it ispresent in a sample to be tested. The sensing unit preferably has anumber of associated attributes including that each of the materialcompositions of the lower binding surface and the upper binding surfacehas properties that are preserved with the use of an effectiveinsulating layer. The tripartite attachment layer may consist of threedistinct materials, or different material compositions of a singlematerial, providing that each element of this layer (the lower bindingsurface, the upper binding surface, and the insulating layer) hasproperties that are suited particularly for its role in the tripartiteattachment layer.

[0017] In a related variation, the upper surface of the tripartiteattachment layer may be used to bind to a ligand layer that is receptiveto or captures a non-specific analyte. That is, the tripartiteattachment layer is not limited to use with an analyte-specific ligandlayer.

[0018] In another embodiment, the sensing unit includes the solidsubstrate of the first embodiment, such as a reflective substrate otherthan a free electron metal (e.g. crystalline silicon), two elements orlayers of an attachment layer, and an analyte-specific ligand layer. Theattachment layer element closest to the silicon surface is comprised of,for example, an organofunctional silial compound. The silane portion ofthe attachment layer forms a covalent linkage to the silicon substratesurface, leaving the reactive organofunctional group available forfurther interaction with a second element of the attachment layer. Thesecond element of the attachment layer is usually an organic polymerfilm that is applied, preferably spin coated, to the organofunctionalsilial compound layer. This organic polymer film provides a controlledenvironment to immobilize the analyte-specific ligand layer or to attachthe non-specific sample to serve as an attachment platform for thenon-specific binding of analyte in the sample.

[0019] A sensing surface may include the solid silicon substrate of thefirst embodiment, a single attachment layer applied using conditionsthat prevent the attachment layer from delaminating from the solidsubstrate, and an analyte-specific ligand layer. The attachment layeris, for example, an organic polymer. This single attachment layerprovides all three functions described in the first embodiment: bindingto the silicon support, immobilizing the ligand layer, and isolating theligand layer from properties of the solid substrate that may inactivatethe analyte-specific ligand layer. The ligand layer captures andimmobilizes a specific analyte when it is present in a test sample.

[0020] Like the tripartite attachment layer, both the dual elementattachment layer and the single attachment layer may be used to bind toa ligand layer that is receptive to a non-specific analyte.

[0021] Detection of the analyte may be improved further through variousmeans of mass enhancement. These mass enhancement techniques typicallyinvolve the secondary binding of an analyte-specific ligand that may ormay not be different from the ligand used to immobilize the analyte onthe surface. This secondary binding ligand may be further linked to anadditional amplification system. These additional amplification systemsinclude, but are not limited to, enzymes (such as horse radishperoxidase or alkaline phosphatase), macromolecules (such as DNA, RNA orferritin) or small particulates (such as polystyrene microspheres, metalsols, silica, self-assembling monolayers or film-forming compounds).

[0022] Each sensing unit is adapted to be held at a defined location ona test piece or slide. Typically, each test piece is able to support apredetermined number of sensing units. The defined positions on the testpiece include marks, bar codes, or other indicia that are useful inidentifying the particular sensing unit on a test piece. The test pieceis of a size and shape to be used with and received by an instrument fordetecting and/or measuring, when present, an analyte or substance ofinterest that is bound to one or more of the sensing units on the testpiece. The instrument includes a compact housing that includes a numberof assemblies or elements. A test piece movement assembly may provideautomatic control of the positioning of the test piece, particularlyrelative to a light beam assembly that is used in detecting whether ornot an analyte of interest is present on the current sensing unit beingtested.

[0023] The instrument may also include a reader assembly for reading themarks or codes on the test piece and regulating the movement of the testpiece using the application of power to a motor in order to properlyposition the test piece and, concomitantly, the sensing unit under test,relative to the light beam assembly.

[0024] The housing includes an aperture for receiving the test piece.The housing may further include a user input assembly, such as a keypad,for requesting information and data related to the detection process.The instrument also has a display unit, for example LCD, that displaysrequested output information, such as the results of the detectionprocess, as well as graphs or plots. The display unit is also useful incommunicating available options to the technician or user related toinformation and data that is available from the instrument. Preferablyalso, the instrument is able to communicate with an external processingsource or computer system that enables the instrument to download dataor other information related to the functions that it performs inconnection with the detection and/or measurement process.

[0025] The light beam assembly includes a source of light that iscontrolled and directed for obtaining information useful in analyzingthe mass of the sensing unit in connection with determining whether ornot the analyte of interest is present. In one embodiment, the lightbeam assembly includes components for providing a number of bounces orreflections of the altered light through the same sensing unit in orderto amplify the changes in the reflected light to improve sensitivity.Such sensitivity is particularly advantageous when the substance ofinterest is present and the mass change of the sensing unit isrelatively small and difficult to detect.

[0026] With respect to further aspects related to the method oroperation for determining whether or not an analyte of interest ispresent, the test piece having a number of sensing units is insertedinto the instrument housing. The test piece movement assembly iscontrolled by a digital controller having a processor so that it ismoved inwardly within the housing until the reader assembly reads a markor code that is interpreted by the processor, and thus, controls thepowering of f of the motor of the test piece movement assembly. Thelight beam assembly provides a beam of light that is controlled andcaused to reflect from the sensing unit being tested. Reflected lightfrom this particular sensing unit is collected and analyzed. In certainembodiments for obtaining light from a sensing unit, the instrument isalso configurable to provide virtually only s-polarized light or,alternatively, p-polarized light. In such cases changes in the detectedsignal due to change in polarization state from a particular sensingunit will be a function of any increase or decrease in the mass of thesensing unit. Preferably, such light is linearly polarized; however,circular or elliptical states of polarization can also be utilized. Theresults of the analysis are stored and available for presentation and/orfor the user to obtain and make decisions based on such results. Thestored information includes not only information related to the resultof whether or not the analyte of interest is present, but alsoinformation related to the identification of the individual test piecesand sensing units.

[0027] In one embodiment of the invention, the system further includes adevice that has a number of assemblies for providing desired functionsassociated with test pieces or test performance. A heating assemblyincludes a number of plates that are heated to enable heat to beconducted to the test piece. The heating assembly also includes asupport frame that permits it and the heated plates to be pivoted foraccess to a number of absorbative members of a humidifying assembly.Each of the absorbative members is soaked with water to enable moistureor vapor to be developed due to the heat generated by the heatingassembly. A mixing assembly is also part of the device for mixing thematerials associated with the sensing units. A barrier manifold having anumber of barrier members is pivotally movable relative to the testpiece when it is positioned within the incubation device. The barriermembers are useful in avoiding cross-contamination among the number ofsensing units with the test piece.

[0028] Based on the foregoing summary, a number of key features of thepresent invention are easily recognized. A system is provided thatincludes a sensing or assay unit comprising an attachment layer thatboth substantially reduces the possibility of delamination of theattachment layer from a substrate and immobilizes a ligand layer that isreceptive to an analyte of interest when it is present with the sensingunit being tested. In one embodiment, the attachment layer is definableas three layers that include an insulating layer for preserving theintegrity and proper functioning of the layers or surfaces that theinsulating layer contacts.

[0029] The system also may include methods and materials for enhancingthe detection and/or measurement of mass change activity of thesubstance of interest, when present. Such mass enhancement techniquesare associated with the sensing unit and assist an appropriateinstrument in its detecting or measuring operations by effectivelyenhancing the presence of the property (mass) being measured.

[0030] The system further includes an instrument for receiving a testpiece having a number of sensing units in order to determine whether ornot one or more of them contains an analyte of interest. The instrumentis compact, easy to use in connection with obtaining desired data orother information and has numerous automatic operational features. Inparticular, the instrument is able to control the position of the testpiece to properly align it with a beam of light to be used for detectionpurposes, conduct the necessary test including analysis of theparticular sensing unit and store the results of the test including anidentification of the particular sensing unit and the time of thetesting. In one embodiment, light is caused to reflect a number of timeson the same sensing unit to increase the sensitivity of the instrument.In other variations, only s-polarized light or p-polarized light isobtained to enhance the sensitivity of the detected signal from asensing unit. The instrument is also able to store and download data toan external computer terminal for additional evaluation or use of theobtained data. Preferably, the system also includes a device for housingthe test piece which is beneficial in controlling temperature, humidityand mixing the materials provided with or as part of the sensing unit.This device includes a barrier manifold that acts to prevent each of thetests samples from contaminating each of the other samples.

[0031] Lastly, an overall sensing system is provided that has all of thenecessary sub-system components for detecting and/or measuring asubstance of interest by relying on mass change activity. Thesesub-system components readily cooperate and function together in meetingsuch main objectives. Each sensing unit is properly formed and preparedfor testing, including use of suitable mass enhancement techniques thateffectively amplify the signal that is detected when the analyte ispresent. The sensing units on the test piece are properly prepared andaccurately positioned relative to the instrument for conducting thedesired test. The instrument itself is highly sensitive to mass activitychange in connection with determining whether or not the analyte ofinterest is present.

[0032] Additional advantages of the present invention will becomereadily apparent from the following discussion, particularly when takeninto account with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 is a diagrammatic representation of a sensing unit thatincludes a tripartite attachment layer;

[0034]FIG. 2 is a diagrammatic representation of the sensing unit ofFIG. 1 illustrating the resultant sensing unit after certain processsteps have been conducted and when the analyte of interest is present;

[0035]FIG. 3 is a diagrammatic representation of the sensing unit ofFIGS. 1 and 2 in which a mass enhancement composition is also utilized;

[0036]FIG. 4 diagrammatically illustrates a particular sensing unit thathas a dual attachment layer;

[0037]FIG. 5 diagrammatically illustrates the sensing unit of FIG. 4after certain process steps have been conducted and in which the analyteof interest is present;

[0038]FIG. 6 diagrammatically illustrates the sensing unit of FIGS. 4and 5 in which a mass enhancement composition is also utilized;

[0039]FIGS. 7 and 8 are flow diagrams related to the making and use ofthe sensing unit of FIGS. 4-6;

[0040]FIG. 9 illustrates an exploded view of an instrument of thepresent invention involved in the detection and/or measurement process;

[0041]FIG. 10 is a block diagram of major components of the instrumentused in analyzing the sensing units and in controlling positioningthereof;

[0042]FIG. 11 is a perspective view of a test piece;

[0043]FIG. 12 is an enlarged, diagrammatic view of the light beamassembly, the test piece movement assembly and the reader assembly usedin an instrument of the present invention;

[0044]FIG. 13 is an exploded view illustrating a number of components ofthe light beam assembly, the test piece movement assembly and the readerassembly of the embodiment of FIG. 12;

[0045]FIG. 14 is a flow diagram setting out major steps related to theoperation of the instrument using one embodiment of a light beamassembly;

[0046] FIGS. 15A-15B are flow diagrams setting out steps related toconducting analyses of a number of sensing units using two test pieces;

[0047]FIG. 16 diagrammatically illustrates a test piece having a numberof sensing units in a desired position under control of the instrumentof the present invention;

[0048]FIG. 17 illustrates another embodiment of an instrument thatincludes a test piece control assembly of a second embodiment;

[0049]FIG. 18 diagrammatically illustrates another view of theembodiment of FIG. 17 in which further details of the test piece controlassembly are shown;

[0050]FIG. 19 is a side elevational view of the embodiment of FIGS. 17and 18;

[0051]FIG. 20 illustrates a housing for enclosing the embodiment ofFIGS. 17-20;

[0052]FIG. 21 is a diagrammatic view of another embodiment of a lightdetection assembly that employs a multi-bounce operation on the samesensing unit in connection with amplifying the reflected lightassociated with determining whether an analyte of interest is present;

[0053]FIG. 22 is a flow diagram setting out major steps related to theoperation of the instrument using the embodiment of FIG. 21;

[0054]FIG. 23 illustrates a perspective view of a device for containingtest pieces that performs a number of functions; and

[0055]FIG. 24 is an exploded view of the device of FIG. 123.

DETAILED DESCRIPTION

[0056] With reference to FIG. 1, an embodiment of a sensing or assayunit 100 of the system of the present invention is schematicallyillustrated. As seen, the sensing unit 100 includes a number of layersmade of different materials. The multi-layered unit 100 provides anumber of functions as will be described later herein. A sample 104 isdeposited on the unit 100. The sample 104 is to be subsequently testedto determine whether or not it contains a specific analyte or substanceof interest. The specific analyte to be tested to determine whether itis present can include any one of a number of different substances,materials and/or macromolecules, such as biological materials,chemicals, peptides and toxins. In one area of applications, the sensingunit 100 is used as part of a testing system and procedure to test forthe presence of unwanted or harmful substances in animal or human bodyfluids. Depending on the results of such testing, appropriate action orsteps can be taken.

[0057] The layers of the sensing unit 100 include a substrate or baselayer 108 that is used as a support member and is made of a materialthat is useful or compatible with the testing instrument and protocol.In a preferred embodiment, the substrate 108 is other than a freeelectron metal (if a substrate has characteristics of a free electronmetal, it is not acceptable and cannot be used), but is preferably madeof a crystalline silicon material that has desired properties for usewith a testing instrument that utilizes reflected light in determiningwhether the analyte of interest is present.

Tripartite Attachment Layer

[0058] The substrate 108 has an attachment layer 112 joined thereto thatis used to immobilize a ligand layer. The attachment layer 112 includesmaterial compositions for performing two main functions. The attachmentlayer 112 must securely attach or join to the substrate 108 and it mustimmobilize, without harm or unwanted alteration, one or more capturelayers including the ligand layer 116. The ligand layer 116 comprises aknown or specific material or macromolecule that properly bonds with theanalyte of interest when it is present in the test sample. The ligandlayer 116 may comprise a number of different materials such asmonoclonal and polyclonal antibodies, antigens, avidin, biotin, nucleicacids, proteins, peptides, and receptors. In a variation of thisembodiment, instead of using ligand layer 116, or any other capturelayer, there is a direct binding of the analyte, when present, to thetripartite attachment layer 112.

[0059] In one embodiment, the attachment layer 112 is tripartite,non-homogeneous and non-porous. Such an attachment layer 112 includes alower binding surface 118, an upper binding surface 120 and aninsulating or intermediate layer 124 disposed between the surfaces 118,120. Unlike known prior art, the tripartite attachment layer 112includes three layers having differing material properties forperforming a number of functions associated with establishing desiredbonding and insulating functions. The lower binding surface 118 ischaracterized by a material composition that causes it to be adequatelybonded to the substrate 108. Such a bonding adequately resistsdetachment or delamination of the lower binding surface 118 from thesubstrate 108. The lower binding surface 118 actively adheres ordisplays enhanced binding attributes to the substrate 108 through one ormore binding mechanisms or techniques, such as adsorption,electrostatic, chemical, covalent or ionic means. Preferred techniquesor means provide a durable and stable lamination of the attachment layer112 to the substrate 108. The upper binding surface 120 preferablyconsists of a material or materials that are different from the materialcomposition of the lower binding surface 118. The upper binding surface120 requires different attributes or properties since its primaryfunction is to provide a proper binding environment for the ligand layer116 so that the ligand layer 116 is not subject to deformation,denaturation, stearic alteration, or partial or total inactivation ofthe ligand(s) that make up the ligand layer 116. Typically, suchmacromolecules require a controlled immobilization environment to retainsuch functional attributes. A failure to retain such attributes resultsin an inability of the ligand layer 116 to bind to a viable analyte ofinterest when it is present.

[0060] The insulating layer 124 is a protective or barrier layer betweenthe lower and upper binding surfaces 118, 120. This layer 124 has twokey functions. The insulating layer 124 prevents unwanted effects oractivities to be transmitted in each of two directions. In a firstdirection, unwanted or potentially harmful effects on the upper bindingsurface 120 due to the material composition of the lower binding surface118 or the substrate 108 are controlled or prevented by the insulatinglayer 124. In a second direction, such effects on the lower bindingsurface 118 or the substrate 108 due to the material composition of theupper binding surface 120 are prevented from occurring. The unwantedeffects involve the transfer of potentially harmful electrostatic,ionic, hydrophobic or covalent properties to one of the two surfaces118, 120 from the other of the two surfaces. The insulating layer 124also prevents the transfer of unwanted effects that may be attributableto the capture layer including ligand layer 116 or any other layer,material or sample that may be included with the sensing unit 100.

[0061] Each of the lower and upper binding surfaces 118, 120 has amaterial or materials composition that is different from the material ormaterials composition of the insulating layer 124. Each of these twobinding surfaces 118, 120, requires properties in order to properlyfunction that are different from the insulating or protective layer. Theinsulating layer 124 may be homogeneous or may be non-homogeneous, suchas including a gradient of different materials. Although the tripartiteattachment layer 112 may be non-porous, or effectively non-porous, andresists permitting liquid to flow through it, the insulating layer 124is the region or section of the tripartite attachment layer 112 thatinhibits such passage of liquid. The lower and upper binding surfaces118, 120 need not be impervious or non-porous. Related properties of theinsulating layer 124 include its ability to resist or opposedelamination forces or activities for relatively long periods of time.

[0062] The compositions of the lower binding surface 118, the upperbinding surface 120 and the insulating layer 124 can include one or moreof different materials or groups of materials that are appropriate inperforming the functions and including the properties required of suchsurfaces and layer. In one embodiment, the substrate 108 includes, forexample, commercially available crystalline silicon that may or may nothave a native oxide layer. The tripartite attachment layer 112 caninclude any combination of, for example, the following materials:inorganic or organic monomeric and polymeric compounds. Examples ofmaterials that can by utilized for the three sections of the tripartiteattachment layer include: 6-azidosulfonylhexyltriethoxy silane,aminoethylamino-propyl trimethoxy silane, aminopropyltriethoxysilane,3-isocyanatopropyltriethoxysilane, phenyltriethoxy-silane,poly(ethylene)glycol, poly(ethylene)oxide, nitrocellulose, paralene,nylon, polyester, polyimides, polyurethane, polystyrene, avidin (and itsderivatives), and biotin, and any combination thereof.

[0063] In certain embodiments, the attachment layer 112 has thefollowing: inorganic monovalent (non-polymeric) compounds and/or organicmonovalent and/or polyvalent compounds that either do not have particlesor do have particles that retain their particle size (do not coalesce)of at least 40 microns and each of these has linear backbone(non-branching, random and/or irregular) structures including, forexample, polystyrene, polyurethane, polyethylene glycol, avidin-biotin,and/or combinations thereof.

[0064] With reference to FIG. 2, a further depiction of the sensing unit100 is provided that illustrates the state of the sensing unit 100 aftera number of assay process steps have been conducted that will bedescribed in greater detail later herein in the context of a secondembodiment of a sensing unit but which steps are also applicable to theembodiment of FIG. 2. Essentially the only substance remaining aftersuch steps is the analyte of interest 106, when present. The analyte ofinterest 106 is bound or immobilized on the accepting or capturingligand layer 116. At this stage of its preparation, the sensing unit 100can be positioned for testing using an appropriate instrument.Alternatively and preferably, as seen in FIG. 3, one or more massenhancement substances or materials 110 used effectively amplify themass change, when present, that is being detected or measured. Thesemass enhancement techniques can take one or more different formsincluding kinetic-active mass enhancement, passive mass enhancement anda self-assembling amplification system. Mass enhancement substances willbe subsequently described in connection with certain examples. Thesemass enhancement substances improve the detection of the analyte ofinterest 106 when it is present and usually involve the secondarybinding of an analyte-specific ligand, which may be further joined toadditional amplification systems.

Dual Element Attachment Layer

[0065] Another embodiment of a sensing unit is schematically illustratedin FIG. 4. This embodiment includes a dual element or laminateattachment layer, instead of a tripartite attachment layer. This dualelement layer, unlike known prior art, is made of different materialsincluding an organofunctional silial compound and an upper element thatattracts or bonds to a desired ligand. This sensing unit 150, like thefirst embodiment, includes a substrate 154 made of a silicon-basedmaterial such as a silicon wafer. The attachment layer 158 includes alower element 162 that has an organofunctional silial compoundincluding, for example, 6-azidosulfonylhexyltriethyoxy silane andaminopropyl-triethoxysilane.

[0066] The lower element 162 has properties like the lower bindingsurface 118 of the first embodiment including primarily providing adurable and stable attachment to the substrate 154. The dual attachmentlayer 158 also includes an upper element 166 that is bound to the lowerelement 162 by one or more of an adsorption, covalent, ionic, chemicaland/or electrostatic attachment. Importantly, the upper element 166 musthave an outer surface 168 that causes a capture layer including a ligandlayer 170 to be bound thereto. In the dual attachment layer embodiment,the outer surface 168 and the remaining portions of the upper element166 are homogeneous. The capture layer including ligand layer 170 hasthe same functions and properties and can be comprised of the samematerial compositions as the capture layer of the first embodiment. Atest sample 174 is provided on the ligand layer 170, similar to that inwhich the test sample 104 is provided on the ligand layer 116 of FIG. 1.

[0067] Like the embodiment of FIGS. 1-3, the sensing unit 150 isprepared for testing whether or not an analyte of interest is present.As seen in FIG. 5, after certain process steps that will be subsequentlydiscussed, the analyte of interest 156 is illustrated as being bound tothe ligand layer 170. At this stage, the sensing unit 150 may bepositioned for testing using, for example, the instrument and variantsthereof that will be described later herein. Preferably, massenhancement systems or substances 160 are provided on the analyte ofinterest 156. Such a mass enhancement system 160 performs the samefunctions that are provided in the embodiment of FIGS. 1-3.

[0068] With regard to further details of the composition and method ofmaking the dual element embodiment, reference is made to FIGS. 7 and 8.As provided in the flow diagrams, together with subsequent Examples,1-3, a number of steps are involved in the making of the sensing unit150. In particular with reference to FIG. 7, at step 200, a crystallinesilicon wafer is obtained. At step 202, the wafer is positioned in aspin-processor for subsequent receipt of the dual element attachmentlayer. Next, at step 206, an organofunctional silial compound, such as6-azidosulfonylhexyltriethoxysilane, is spin coated onto the wafer. Atstep 208, an organic polymer, such as polystyrene, is next spin coatedonto each lower element 162 to form each upper element 166. As describedat step 212, the wafer and the lower and upper elements 162, 166 arethen baked at a sufficient temperature for a suitable time period inorder to cure them.

[0069] As noted at step 214, the wafer is cut into strips to form eachdual element attachment layer 158 that includes the lower and upperelements 162, 166.

[0070] In accordance with step 216, a number of sensing units are thenmounted on a glass slide. At step 218, a masking step is performed bywhich each sensing unit is separated from the others in a manner thatreduces potential contamination between or among the sensing units, aswell as such placement and separation of the sensing units beingcompatible with the placement and location of identifying bar codes orother indicia.

[0071] The dual element attachment layer 158 may be tested for stabilityat step 220 but need not be. In particular, the attachment layer 158 maybe tested to determine its ability to resist or oppose delaminationforces or activities.

[0072] At step 222, a primary ligand layer 170 is coated to the outersurface of each sensing unit 150. As set out at step 224, the dualelement attachment layer 158 and accompanying ligand layer 170 are thenbaked at a determined temperature for a sufficient time in order toincubate the primary ligand layer 170. Next, at step 226, any additionalprimary ligand binding sites in each sensing unit are blocked to preventunwanted, non-specific binding. Such blocking is usually preceded by arinsing and drying of each sensing unit 150.

[0073] With reference to FIG. 8 at step 230, a test sample is thendeposited onto each sensing unit 150. The primary ligand layer 170comprises a known or specific molecule that properly bonds with theanalyte of interest when it is present. After adding the test sample, afurther incubation is conducted at step 234 in order to allow specificbinding of the analyte, when present, to occur.

[0074] About the time the testing or assay is to be performed, theblocking composition is rinsed away and, preferably, a secondary ligandis added, in accordance with step 238. At step 242, each sensing unit150 is once again incubated including, in this embodiment, the primaryand secondary ligands and an analyte of interest. As previously noted,it is typically beneficial to include a mass enhancement composition.Hence, at step 246, after the incubation and secondary ligand binding, afurther rinsing is conducted and then a mass enhancement composition isadded, such as an enzyme substrate. Now each sensing unit 150 is readyto be utilized with a detecting or measuring instrument to read ordetect any change in mass in the particular sensing unit 150 due to theanalyte of interest, when present, at step 250.

[0075] Although the foregoing description relates to the method ofmaking the dual element attachment layer, such steps are equallyapplicable to a process for making the tripartite attachment layer,except where differences may arise due to the tripartite nature of thetripartite attachment layer, as compared to the dual element attachmentlayer.

[0076] Further detailed explanations of the steps denoted in FIGS. 7 and8 are provided in the following Examples in which Example 1 relatessubstantially to the steps of FIG. 7 and Examples 2 and 3 relate to thesteps of FIG. 8 and some of the steps of FIG. 7.

EXAMPLE 1

[0077] A batch of monocrystalline silicon wafers was obtained havingpolished silicon wafer surfaces with surface native dioxide thicknesses.The wafers were then positioned in a spin-processor for receipt of thedual element attachment layer.

[0078] A 2% 6-azidosulfonylhexyl triethoxysilane (azidosilane) insolution of 95% 200 proof ethanol and 5% dH₂O with 1 drop 1N sodiumhydroxide was prepared. The azidosilane solution was spin coated ontothe silicon wafer by placing a 300 μl sample of the azido-silanesolution in the center of the silicon wafer while the wafer was spinningat 5,000 rpm in a dry N₂ environment. The azido-silane coated wafer wasthen spun dry, spin rinsed with 1 mL of 200 proof EtOH, and baked at110° C. for 10 minutes.

[0079] A 0.5% 212,000 MW styrene (30-33 mV) in toluene solution wasprepared. The styrene solution was spin coated onto the azido-silanelayer by placing a 500 μl sample of the styrene solution in the centerof the azidosilane coated wafer while the wafer was spinning at 5,000rpm in an ambient environment. The dual element attachment layer andwafer were then baked at 100° C. for 18 to 20 hours and the attachmentlayer was then checked for attachment stability.

EXAMPLE 2

[0080] Rabbit anti-Bacillus globigii polyclonal antibodies are dilutedto 9 μg/mL in 50 mM HEPES buffer (pH 8.0), and 15 μL of this dilutedcapture antibody are dispensed onto each sensing unit in the test piece.The test piece is then incubated for one hour at 37° C. under highhumidity conditions. After rinsing with distilled water and drying thetest piece with compressed nitrogen gas, any additional protein bindingsites in the sensing units are blocked by adding 18 μL StabilCoat™ toeach sensing unit. After incubation for one hour at 37° C., excessliquid is removed by aspiration, and the test piece may be stored at 4°C. until use.

[0081] When the assay is to be carried out, the excess blocking proteinis rinsed away using distilled water. Fifteen microliters of sample(diluted into Bacillus globigii Sample Buffer consisting of 50 mM HEPES,pH 8.0, with 0.2% bovine serum albumin, 0.01% Tween®-20) are dispensedinto the sensing units, and the test piece is incubated at 37° C. forone hour. The sensing units are then rinsed with distilled water anddried with compressed nitrogen gas.

[0082] In the next step, 15 μL biotinylated goat anti-Bacillus globigiipolyclonal antibodies (diluted to 2 μg/mL in anti-Bacillus globigiiSample Buffer) are dispensed into each sensing unit. The test piece isincubated for thirty minutes at 37° C. The reaction is stopped byrinsing the test piece with distilled water and drying with compressednitrogen gas. Next, 10 μL streptavidin/polymerized horseradishperoxidase conjugate (Sigma Chemical Company; St. Louis, Mo.), diluted1:6000 in Bacillus globigii Sample Buffer, are added. The test piece isincubated for thirty minutes at 37° C., and the test piece is thenrinsed with distilled water and dried with compressed nitrogen gas.

[0083] Finally, 10 μL TMB Reaction Substrate (Kirkegaard PerryLaboratories; Gaithersburg, Md.) are added to each sensing unit. Afterincubation for 15 minutes at 37° C., the reaction is stopped by rinsingthe test piece with distilled water. The test piece is dried withcompressed nitrogen gas, and the amount of reaction product is measuredon the instrument.

EXAMPLE 3

[0084] Sensing units in the test piece are coated with 15 μL of rabbitanti-Listeria polyclonal antibodies, diluted to 23 μg/mL in PBS (10 mMphosphate buffer, pH 7.4, containing 150 mM NaCl). After incubating thetest piece for one hour at 37° C. under high humidity conditions, thetest piece is rinsed with PBST (PBS containing 0.05% Tween®-20),following by rinsing with distilled water. The test piece is then driedwith compressed nitrogen gas. Any additional protein binding sites areblocked by adding 20 μL bovine serum albumin, 30 mg/mL in PBS, to eachsensing unit. After incubation for 30 minutes at 37° C., excess liquidis removed by aspiration, and the test piece may be stored at 4° C.until use.

[0085] In the next step, 15 μL sample, consisting of heat-killedListeria in media, is added to the sensing units. The test piece isincubated for 30 minutes at 37° C., and then rinsed with PBST anddistilled water. The test piece is dried with compressed nitrogen gas.Next, 15 μL rabbit anti-Listeria polyclonal antibody/horseradishperoxidase conjugate (Kirkegaard Perry Laboratories), diluted 1:500 inBiostride™ conjugate diluent, are added to each sensing unit. The testpiece is incubated for 30 minutes at 37° C., rinsed with PBST anddistilled water, and then dried with compressed nitrogen gas.

[0086] Finally, 15 μL TMB Reaction Substrate (Kirkegaard PerryLaboratories) are added to each test well. After incubation for 15minutes at 25° C., the reaction is stopped by rinsing the test piecewith distilled water. The test piece is dried with compressed nitrogengas, and the amount of reaction product is measured on the instrument.

Instrument and System Operation

[0087] With reference to FIGS. 9 and 10, the system for detecting aspecific substance or analyte of interest also includes an instrument300 for obtaining and analyzing data related to the determination ofwhether or not the analyte of interest is present with a sensing unit100. The instrument 300 includes a housing 304 for containing assembliesand components utilized in the detection and analysis process. Thehousing 304 is characterized by its compact size and relatively smallfootprint. Its compact size is achieved by specific selection andarrangement of such assemblies and components within the housing 304.The housing 304 includes a lower containing unit 308 having a number ofwalls and an upper cover 312 that is connected to the lower containingunit 308. The cover 312 has an upper face that includes an input unit orkeyboard 316 by which the technician or user can input informationincluding requests for data to the instrument 300. The upper face alsohas a window 320 for receiving a liquid crystal display (LCD) 352 (FIG.10) that is useful in displaying menus or other information forselection, together with results of the analysis conducted by theinstrument 300, such as graphic plots of data related to the detectionprocess. An end wall 324 of the lower housing unit 304 has a receiverslot 328 formed near the bottom of this end wall 324. The receiver slot328 is configured and of a size to receive a test piece or slide 332. Asseen in FIG. 11, the test piece 332 is an elongated, relatively flatmember that is able to hold a number of sensing units 100, each of whichis spaced from any other such unit 100. The test piece 332 has indicia334 for indicating the location and/or identity of the particularsensing unit 300. That is, such indicia 334 may include one or moremarks associated with or located at each sensing unit 100 a-100 l.Consequently, when such a mark is read or detected by the instrument300, this read information can be used to initiate the analysis anddetection process. Additionally or alternatively, such indicia 334 mayinclude identification information, such as in the form of a bar codeassociated with or located adjacent to each sensing unit 100 a-100 l.The bar code for each sensing unit represents the identity of theparticular sensing unit 100 a-100 l. Accordingly, such bar codeinformation can be used to maintain or record identity information forthe particular sensing unit that is being tested and distinguish it fromother sensing units.

[0088] Returning to FIGS. 9 and 10, one of the assemblies of theinstrument 300 is a controller assembly 340. The controller assembly 340has a number of electronic components, including a digital controller344 that is responsible for controlling and interacting with a number ofdevices or elements. The digital controller 344 includes one or moreprocessors that are involved with determining whether or not a specificanalyte of interest is present in the sensing unit 100 being testedusing data that the processor receives from other components of thecontroller assembly 340. The digital controller 344 communicates with aLCD controller 348, which is used in controlling and providing thepresentation of information by the liquid crystal display (LCD) 352,under the ultimate control of the digital controller 344. Similarly, thedigital controller 344 communicates with the keypad 316 through a keypadinterface 356 before sending such information to the digital controller344. The controller assembly 340 also includes memory storage forstoring data and executable code. In that regard, a program memory 360and a data memory 364 communicate with the digital controller 344. Theprogram memory 360 stores programmed code or algorithms for analyzingthe data-related signals that are received by the digital controller344, including those related to detecting the presence of an analyte ofinterest. The data memory 364 is utilized to store results associatedwith the detection process, such as values related to the results of thedetection process.

[0089] Returning to FIG. 9, the components and assemblies of thecontroller assembly 340 are mounted on one or more printed circuitboards 368 that are sized to be contained within portions of the lowercontaining unit 304. Also included within the lower containing unit 304and in communication with the digital controller 344 is a number of dataproviding/generating or peripheral assemblies, with at least portionsthereof disposed below the printed circuit board(s) 368. Morespecifically, a test piece control assembly 372, a reader assembly 376and a light bean assembly 380 are provided, with at least some elementsof each of these three assemblies being fixed to a mounting plate 384.

[0090] With reference to FIGS. 12 and 13, as well as FIGS. 9 and 10,each of these assemblies will be described in greater detail. The testpiece control assembly 372 is employed in connection with controllingmovement of the test piece 332 having a number of sensing units 100 thatmay have an analyte of interest. The test piece control assembly 372includes a motor 388 that is held or supported by a motor mount 392.Operation of the motor 388 including providing and removing powerthereto is accomplished using a motor controller 396 that communicateswith the digital controller 344. When it is appropriate to move the testpiece 332, the digital controller 344 generates the necessary signal forreceipt by the motor controller 396 in order to generate a suitablesignal for the motor 388. The output of the motor 388 is connected to afirst gear 400 that rotates when the motor 388 is powered. The firstgear 400 operatively engages the second gear 404. The two gears 400, 404are at right angles to each other to contribute to the compact size ofthe instrument 300. Rotation of the first gear 400 causes the secondgear to also rotate. The second gear 404 is connected to a drive shaft408 having a first collar 412 adjacent to the second gear 404 and afirst bushing 416 spaced from the collar 412 along the drive shaft 408.First and second support arms 420, 424 receive and support the driveshaft 408. Between the support arms 420, 424, the shaft 408 traverses atest piece path 428 defined in the mounting plate 384. The first andsecond support arms 420, 424 are held to the mounting plate 384 onopposite sides of the test piece path 428. Inwardly adjacent to each ofthe two support arms 420, 424 is first drive wheel 432 and second drivewheel 436, respectively. Each of these two drive wheels 432, 436 istoothed or otherwise configured to satisfactorily engage edges or otherportions of the test piece 332. Each of the two drive wheels 432, 436,through engagement with the drive shaft 408, rotates when the driveshaft 408 is driven or rotated to thereby cause the test piece 332 tomove along the test piece path 428. To complete the discussion of theelements supporting the drive shaft 408, a second bushing 440 isprovided about the shaft 408 adjacent to the second drive wheel 436 anda second collar 444 is disposed about the drive shaft 408 on theopposite side of the second support arm 424. The test piece controlassembly 372, as seen in FIG. 4, also includes a back plate 448 havingan acceptor opening 452 oriented with the receiver slot 328 formed inthe end wall 324 of the lower containing unit 308 in order to receiveand guide the test piece 332 along the test piece path 428.

[0091] With regard to positioning the test piece 332 at a desiredposition for a particular sensing unit 100 to be analyzed, the readerassembly 376 is utilized. The reader assembly 376 reads or obtainsinformation using the indicia 334 on the test piece 332. This obtaineddata is sent to the digital controller 344, which analyzes suchinformation in controlling the application of power to the motor 388. Asseen in FIGS. 12 and 13, the reader assembly 376 includes a readerelement 460 that is precisely and accurately positioned along the testpiece path 428 to read indicia 334 on the test piece 332. The positionof the reader element 460 is precisely positioned relative to certainelements of the light beam assembly 380, as will be understood morefully when a more detailed description of the light beam assembly 380 isprovided. The reader element 460 communicates its output to processingcircuitry 464 of the controller assembly 340, as illustrated in FIG. 9.The processing circuitry 464 processes the signal received from thereader element 460 to output a reader signal, including providingdesired amplification, filtering to obtain the proper frequency for thesignal and removing unwanted noise from the signal. When a modulatedlight source is utilized, filtering that enhances the ability todifferentiate the desired signal from an unwanted DC level of noise isprovided. In such a case, such filtering can include the use of Fouriertransform techniques that are useful in extracting the DC noise portionfrom the input signal, then applying an electronic filter andsubsequently integrating the resultant signal to obtain the desired,detected signal. After such processing, the processed reader signal isapplied to a decoder 468. In the case of reading a bar code, the decoder468 includes logic for deciphering or otherwise analyzing the signalinput thereto in order to generate a digital signal that is indicativeof the bar code currently read by the reader element 460. This digitalor decoded signal is applied to a serial interface 472 that communicateswith the digital controller 344 to thereby provide test piece 332position information to the digital controller 344.

[0092] Returning to FIG. 12, the reader assembly 376 also includes areader mount 476 having a hole for receiving and tightly engaging thereader element 460. The reader mount 476 is adjustable relative to thetest piece path 428 in order to properly align it relative to the testpiece path 428. In that regard, the reader assembly 376 also includes anadjusting element 480 located in an adjusting slot 484 whereby thereader mount 476 can be variably located relative to a reader support488 that is fixably held to the mounting plate 384 adjacent to the testpiece path 428.

[0093] The light beam assembly 380 also has elements that must beaccurately and precisely located relative to the test piece path 428.The light beam assembly 380 provides a light beam that is used inilluminating the sensor containing the analyte of interest, whenpresent, on the particular sensing unit 100 when the test piece 332 iscontrollably stopped along the test piece path 428 in order to enablethe light beam to strike and reflect from the sensing unit 100. In theembodiment of FIGS. 9, 10, 12 and 13, the light beam assembly 380includes a laser module 496 that provides a source of monochromaticlight, although a non-laser source of generated light is feasible. Themonochromatic light source may include properly filtered white light, alight emitting diode (LED) or a laser diode. The application of powerand the control associated with the light beam outputted by the lasermodule 496 is provided using the digital controller 344 and a laserregulator 500, which communicates with the digital controller 344. Asillustrated in FIGS. 7 and 8, the laser module 496 is supported in anoptics mount 504. In the embodiment of FIG. 8, the light beam assembly380 also includes an adjustable or rotatable linear polarizer 508 fordesirably preparing the polarization state of the light beam that is tobe incident upon the sensing unit 100 after passing through a polarizerplate 512. More specifically, the linear polarizer 508 is orientated tooutput only the “s” component or “s” polarization of the light itreceives while essentially eliminating or minimizing the “p” componentor p-polarization of the light it receives. In another embodiment, awave plate or compensator is also used in combination with the polarizer508 prior to the light striking the sensing unit 100 in order to assistin achieving the desired polarized state of the light. In otherembodiments, circular or elliptical states of polarized light areobtained and utilized.

[0094] The incident linearly polarized light from the polarizer plate512 is directed to the particular sensing unit 100. The incident lightstrikes a small area or point on the sensing unit including the analyteof interest, when present. The reflected light includes usefulinformation in connection with determining a change in mass when thespecific analyte of interest is present. The light beam assembly 380also includes a detection assembly 520 that is properly positioned andaligned to receive such reflected light. As long as metal reflectors ormaterials that have a large imaginary component (e.g., greater than0.03) of index of refraction are not being used as part of the sensingunit 100, mostly linearly polarized light (s-polarization only) willleave the sensing unit 100 after reflection.

[0095] In other embodiments, the light beam assembly 380 is configuredto result in only p-polarized light after reflection. An ellipticalstate of the polarized light can also be achieved in which theelliptical polarized light strikes the sensing unit 100 and theresulting polarized state of reflected light is also elliptical.Alternatively, circular polarized light may be utilized. However, thecircular polarization may undergo an interaction at the sensing unit 100that changes the polarized state of the reflected light to be slightlyelliptical.

[0096] The detection assembly 520 includes a detector polarizer 528 thatremoves, extinguishes, filters or absorbs the linearly polarized lightthat it receives. In another embodiment, a wave or compensator plate maybe optionally used with the polarizer 528. Such a component assists infiltering the reflected state of polarized light of any small componentof p- or s-polarized light provided in the reflection from the sensingunit 100. It is possible that the composition of the sensing unit 100may cause some ellipticity to be present in the reflected light and/orthat the ellipticity is caused by slight imperfections or smallmisalignments of the polarizing components. After the foregoing opticalprocessing, the only signal produced, in view of the removal of thelinearly polarized light by the detector polarizer 528, will be due toan increase or decrease in the mass of the sensing unit 100. It has beenobserved that this arrangement decreases the sensitivity to smallincreases in sensing unit mass changes, with the sensitivity beingmainly due to the use of only s-polarized light. On the other hand, adecrease in sensitivity is experienced when other than p- or s-polarizedlight enters the detector polarizer 528. If any p-polarized light werepresent, there would be a lower signal-to-noise ratio, which means lesssensitivity to small mass changes. More particularly, if the imaginarycomponent of the index of refraction of the sensing unit 100 is at least0.05, then the light leaving the sensing unit will be ellipticallypolarized. Elliptical polarization has both “s” and “p” componentspresent. When both of the components are present, the light cannot beextinguished by the detector polarizer 528. In the embodiment beingdescribed, the s-polarized light is extinguished and the p-polarizedlight would continue along the optical detection path. This p-polarizedlight constitutes optical noise and decreases the sensitivity of theinstrument.

[0097] In the embodiment of FIGS. 12 and 13, the detection assembly 520also includes a compensator wave plate 524 that is optically positionedbetween the sensing unit 100 and the detector polarizer 528. Thecompensator wave plate 524, typically a quarter-wave plate, allowselliptically polarized light that might leave the sensing unit 100 to beconverted to plane or linearly polarized light having only the “s”component. When linearly polarized light is present, it can then beextinguished by the detector polarizer 528 prior to detection of anylight signal that might be generated due to a mass or thickness changein the sensing unit 100.

[0098] It should be appreciated that the wave or compensator platepreviously discussed can be used on either side of the reflection fromthe sensing unit 100. The choice of retardation in the compensator plateis based on what is required to optimize the signal-to-noise ratio ofthe particular optical component configuration. Depending upon suchcircumstances, a ⅛, ¼, ½ or other wave retardation can be selected.

[0099] In another embodiment, instead of s-polarized light being appliedto the detector polarizer 528 to be extinguished by it, only p-polarizedlight is produced and applied to the detector, with the “s” componentpolarizer 528 already having been essentially eliminated or minimized bythe polarizer elements that output the incident light to the sensingunit 100.

[0100] The output of the detector polarizer 528 is applied to a detectorunit 532 that includes, for example, a photodiode for use in detectingor measuring the intensity of the light received by it. The intensity ofthe light detected relates to the change in mass due to the analyte ofinterest when it is present with the sensing unit 100 as will bedescribed further herein. As seen in FIG. 10, the detector unit 532communicates with detector processing circuitry 536 of the controllerassembly 340 (FIG. 9). The detector processing circuitry 536 processesthe analog signal from the light detector unit 532 including appropriateamplification and signal filtering including, as previously noted, theuse of Fourier transform filtering. The output of the detectorprocessing circuitry 536 is applied to an analog-to-digital converter(ADC) 540 for converting the analog signal to a digital signalacceptable to the digital controller 344. This digitized light signalrepresents the light intensity of the reflected light from the sensingunit 100, including analyte of interest, when present. This digitallight signal is then analyzed in connection with making a determinationregarding the presence or absence of the analyte of interest with theparticular sensing unit 100 being tested.

[0101] In another embodiment, instead of a photodiode type of detection,an imaging detector, such as a video type, charge coupled device (CCD)and so forth, can be used to capture intensity change of the sensingunit as well as an image of the sensing unit being tested. This type ofimage inspection would be equivalent to scanning the sensing unit with a0.012 mm laser beam. Using data from about 0.012 mm section of a sensingunit, different types of digital analysis can be applied to determinethe amount of material captured. For instance, if some non-specificmaterial were also captured with the analyte of interest, and they werelarger in size or had different polarization properties, their presencecould be detected when their size is in some reasonable relation to the0.012 mm detection resolution, or the polarization property were highenough above general noise. This means that these areas are potentiallyselectable and can be eliminated from the overall signal.

[0102] In another embodiment, an optical apparatus involved with thecontrol of the incident light and collection of reflected light utilizestwo detectors and no compensator plate. Such a configuration does notcontain any moving or adjustable components like polarizers orcompensator plates. In accordance with this configuration, light willreflect from the sensing unit at the proper or desired angle other than0°, 90° or Brewster's angle. After leaving the sensing unit, the lightwill again reflect from a detector or silicon substrate that ispositioned at or near Brewster's angle. This reflection will eliminateall of the p-polarized light and leave only s-polarized light. The useof a silicon detector for the reflected light after the sensing unit isbeneficial since a reading of the amount of p-polarized light can beobtained, which may prove valuable in making precise calculationsregarding the material on the surface of the sensing unit. The remainderof the reflected light will be collected by a final or another detector,comparable to those previously discussed. The final value of thes-polarization light that was collected can be compared tos-polarization light that is collected from a sensing unit that has noanalyte of interest. An instrument that includes this optical apparatuscan have three outputs, s-polarized light, p-polarized light and theratio of the p-to-s amplitudes. This ratio is a common ellipsometriccalculation and can assist calculating other values of interest.

[0103] Additional details of the embodiment of FIGS. 12 and 13associated with the analysis are next described with reference to theflow diagram of FIG. 14. In accordance with the method of thisembodiment, unlike known prior art, only one polarized light componentis received at the detector polarizer 528, such as the s-polarization,while the linear polarizer elements 508, 512 and the detector polarizer528 remain stationary in their same position or orientation that eachhad when the incident light is first generated and then directed to theparticular sensing unit 100 that is being tested.

[0104] With respect to using the determined light intensity from thesensing unit 100 in order to detect whether or not the analyte ofinterest is present, the present invention relies on previouslygenerated standardized data that may be presented in the form ofstandardized curves or plots. Such standardized data may also berepresented by a number of discrete data points stored in memory thatcan be interpolated to arrive at any value between such discrete datapoints. Such data points relate to values of light intensity associatedwith a number of standardized or accurately measured masses for a numberof sensing units that include masses that are intended to correlate withchanges in mass to a sensing unit currently being tested when asubstance of interest is present. For example, the instrument 300 isused with a standardized sensing unit 100 that includes a massrepresentative of a mass when an analyte of interest is present.Measurements are conducted using this standardized sensing unit. Data iscollected for this particular sensing unit having this first known mass,as well as other sensing units having other known masses. Such knownmasses are correlated with light intensity values that are obtained. Theresults of such standardization development include correlated massesand light intensity values, as noted in step 550 of FIG. 14.

[0105] With such information or data available, step 554 defines acalibrating step that is conducted prior to performing the detectionprocess associated with a particular sensing unit 100. Specifically, thedetector linear polarizer 528 is calibrated by desired rotation thereofto obtain a reference light intensity that passes through it when aknown or standard sensing unit is present to reflect light. Thisreference light intensity may have a zero or substantially zerointensity value. After this calibrating step that involves rotating thedetector linear polarizer 528, at step 558, a sensing unit 100 is thenproperly positioned to receive incident light. At step 562, the incidentlight is directed to a point or small area on the particular sensingunit 100. Reflected light from this point on the sensing unit 100,including analyte of interest when present, is received through thecompensator wave plate 524, in accordance with step 566. At step 570,the reflected linearly polarized light is received through the detectorpolarizer 528, which is not rotated during this detection process butremains stationary, as recited in step 574. After the intensity of thelight is obtained using the light detector unit 532, the detectorcircuitry 536 and the analog-to-digital circuitry 540, the digital lightsignal representative of the intensity is applied to the digitalcontroller 344. At step 578, the digital controller 344 including aprocessor thereof compares the digital light signal with the previouslydeveloped data, as noted in the discussion of step 550. Based on acomparison between the previously developed standard data and theobtained light signal, a determination is made as to any change in massin the sensing unit 100. Based on such a determination, a result can beprovided as to whether or not the analyte of interest is present.

[0106] The result of this determination can be displayed using theliquid crystal display 352. Additionally or alternatively, such resultinformation, as well as other data, can be supplied or downloaded to anexternal apparatus, such as a computer system using a serial I/Oconnection 590 of FIG. 10 that communicates with the serial interface472 to the digital controller 344. The data associated with the resultof each such test can also be stored in the data memory 364 for lateraccess and use.

[0107] Regarding data that is obtainable in connection with analysis ofone or more sensing units 100, a further description is provided withreference to FIGS. 15-16. In accordance with this method, a differentialanalysis is conducted. More specifically, a difference in results isobtained between related sensing units 100 found on successively testedtest pieces. That is, a difference is taken between the results obtainedfor a first sensing unit, which is being tested for an analyte ofinterest, and a sensing unit that does not have such an analyte ofinterest. The first sensing unit is positioned on a first test piece andthe second sensing unit is positioned on a second test piece at acorresponding location. The difference that is found is a signal orlight intensity due to the analyte of interest when present from thechemical reaction or capture process. These operational steps alsodiffer from known prior art in connection with the use of a controllablemotor for automatically positioning each of a number of sensing units ona test piece relative to components that are involved in the detectionprocess.

[0108] FIGS. 15A-15B illustrate flow diagrams setting out major steps inconjunction with obtaining results and associated data for a number ofsensing units 100 on a first test piece 332 for use in determiningwhether one or more of them has an analyte of interest. In accordancewith step 600 of FIG. 15A, a first test piece 332 having a number ofsensing units is positioned for movement relative to the instrument 300.At step 604, the first test piece 332 is moved relative to theinstrument 300 along the test piece path 428. A continuous check is madeat step 608 for a mark and/or identification code (bar code) or anyother indicia on the first test piece indicative of a first sensing unit100. At step 612, movement of the first test piece 332, under control ofthe test piece control assembly 372 and the controller assembly 340, isdiscontinued, based on a determination using the reader assembly 376that the first sensing unit 100 on the first test piece is properlypositioned for analysis, including the obtaining of a reading related towhether or not an analyte of interest is present with this first sensingunit 100. A desired position of the test piece 332 along the test piecepath 428 for conducting the test on the first sensing unit 100 a isillustrated in FIG. 16. Subsequently, the light beam assembly 380 isactivated to take a reading and conduct the analysis for this firstsensing unit of the first test piece at step 616. The reading obtainedis indicative of the presence or absence of the analyte of interest forthis particular sensing unit 100 and at step 620, the results andassociated information for this first sensing unit on the first testpiece 332 is stored in the data memory 364. In one embodiment of theinvention, the data that is stored includes a location number indicativeof the position or number of the sensing unit 100 for the first testpiece 332, an identification code that identifies the sensing unit, theresult of the analysis in terms of a quantitative value obtained usingthe digital light signal generated using the light beam assembly 380 andsubsequent processing circuitry, the date that the analysis wasconducted and the time of such analysis. In accordance with step 624,steps 604-620 are repeated for each sensing unit 100 that is found withthe first test piece 332. Then a second test piece 332 having acorresponding number of sensing units 100 is provided, with each ofthese not having an analyte of interest. Steps 600-624 are conductedusing this second test piece 332 in accordance with step 628. Withrespect to the results that are obtained using the digital light signal,a difference is taken between corresponding sensing units of the firstand second test pieces when there are matching identification codes atstep 632 of FIG. 15B. For example, the determined result of the analysisfor location 1 of the first test piece is subtracted from the determinedresult for location 1 of the second test piece when the identificationcodes match and these two locations correspond to each other. As noted,any sufficient difference is indicative of a mass or thickness changeand, concomitantly, an indication that an analyte of interest is presentwith the sample on the sensing unit 100 that was tested. At step 636,the result of each of the subtractions is saved or stored and, if theresult is less than zero, the value 0.0 is stored. At step 640, on theother hand, if it is found that there is a lack of correspondencebetween the first and second test pieces, no subtraction is taken and noreadings are stored.

[0109] The program code or software that is useful in implementing theforegoing steps also includes a number of capabilities, which areidentified by steps 644-652 of FIG. 15B. At step 644, a recall menu isdisplayed using the liquid crystal display 352. A number of recallfunctions are available for implementation. “Recall Last”, when invokedor pressed, displays information about the last test that was conductedand the reading taken including displaying identification information,the date and time that the reading was obtained, together with thereading result that, in one embodiment, is displayed in volts.“Recall/Scroll”, when invoked or pressed, displays the same results as“Recall Last” and scroll keys are used to enable the user to scroll overand back through all memory locations. “Recall By ID”, when invoked orpressed, allows the user to enter the identification code andinformation is displayed relating to all readings that have thatidentification code.

[0110] At step 648, the user is able to enter a “Plot Request” by whicha number of samples of data for a single identification code aredisplayed. In order to make this request, the desired identificationcode is entered and an initial number of readings, up to a maximumnumber, is plotted and displayed. When there are more than the maximumnumber of readings for the entered ID, the additional or excess samplescan also be plotted by pressing an appropriate key on the keypad 316.When there is no match between the requested identification code andavailable readings, a display is provided to the effect that no suchreadings are available for that entered identification code.

[0111] In accordance with step 652, the program code includes a numberof utilities that can be accessed. Display 352 is used to provide theutilities menu or menus. The utilities menu includes “Show System” fordisplaying current information related to the particular instrument 300including: the current memory location for the sensing unit under test;the total number of memory locations for the particular instrument; thenumber of memory locations still available; the current date; thecurrent time; the current identification code; the number of locationsthat will be read associated with a test piece having the sensing units;the current gain for the detector processing circuitry 536; the currentsoftware version and the current hardware version. The utilities menualso includes a “download data” which is used to download all currentdata stored in the data memory 364. The downloaded data can be receivedby, for example, a personal computer. The utilities menu also includesthe following: “change identification code” by which the user is able tochange the current identification number or code to another number orcode; a “change gain” by which the user is able to adjust the gainbetween minimum and maximum values; “change number of locations” bywhich the user is able to adjust the number of locations on the testpiece 332 that will be automatically read; “laser on/off” by which thepower to the laser can be separately controlled; and “motor CW/CCW” bywhich a key is used to turn on the motor 388 and toggle the direction ofthe motor in connection with confirming the current direction thereof.Numerous other utilities can be provided or implemented depending uponthe needs of the user.

[0112] In another embodiment of an instrument primarily characterized bya differently configured test piece control assembly 660, reference ismade to FIGS. 17-20. The test piece control assembly 660 for moving thetest piece 332 includes first and second drive rollers 662 a, 662 b. Adrive plate 664 is operatively connected to each of the two driverollers 662 a, 662 b. A continuous conveyor belt 668 is disposed overthe drive rollers 662 a, 662 b, as well as the drive plate 664. The testpiece control assembly 660 also includes a guide assembly 670 that isuseful in properly locating or guiding the test piece 332 during itscontrolled movement along and on top of the conveyor belt 668. In thatregard, the drive roller 662 a preferably has a biasing mechanism, suchas a spring, that causes desired movement in a direction toward theguide assembly 670 to provide suitable positioning of the test piece 332relative to the guide assembly 670. More specifically, the guideassembly 670 includes a number of guide rollers 674 that are spacedalong the length of the conveyor belt 668. In the illustratedembodiment, four such guide rollers 674 a-674 d are provided. Each ofthe free ends of the guide rollers 674 engages the edge of the testpiece 332. The guide assembly 670 also includes a pair a pawls 680 a,680 b that are located adjacent to the entry end of the test piece 332onto the conveyor belt 668. Each of the two pawls 680 a, 680 b isdesirably positioned for guiding the test piece 332 relative to the topof the conveyor belt 668 as the test piece 332 is received. The testpiece control assembly 660 also includes first and second support walls682 a, 682 b that are spaced from each other and have the drive rollers662 a, 662 b, together with the drive plate 664 and the conveyor belt668, positioned therebetween. The support wall 682 a supports the guiderollers 674 and the pawls 680 relative to the conveyor belt 668 and thetest piece 332 when it is present in the instrument. As illustrated inFIG. 17, the embodiment of FIGS. 17-20 also includes the support walladjustment assembly 684. This adjustment assembly 684 is operablyconnected to the second support wall 682 b and is used in moving oradjusting the position of the second support wall 682 b relative to thefirst support wall 682 a. Accordingly, the spacing or distance betweenthe two walls 682 a, 682 b can be varied in accordance with the width ofthe test piece 332. In this way, an optimum spacing is achieved that isa function of the width of the test piece 332 so that the test piece 332can be properly located and guided between the support walls 682 a, 682b. As further denoted in FIG. 17, this embodiment also includes a lightbeam adjustment assembly 686 that includes an alignment member 688 and anumber of fasteners 690 joined thereto. The fasteners 690 are operablyconnected to the laser module 496 of the light beam assembly 380.Optimum positioning of the laser module 496 and, concomitantly, optimumdirecting of the light beam outputted therefrom is achieved byadjustment of the position of the laser module 496 using the fasteners690. By this arrangement, instead of time-consuming and potentiallyimprecise locating of the laser module 496 in the instrument, adjustmentto obtain its optimum position is achieved by movement of the fasteners690 causing desired movement of the laser module 496 until the optimumposition is found. With reference to FIG. 20, a housing 692 for thisembodiment is illustrated. The housing 692 includes an angled receiverslot 694 that is geometrically designed to facilitate the entry of atest piece 332 so that it is properly guided for receipt by the testpiece control assembly 660. The housing 692 also has a pair of cut-outs696 a, 696 b that permits manual access and movement of the test piecerelative to the mechanisms and components located within the housing692.

Additive Polarization Optics

[0113] Other embodiments of the assemblies and components of theinstrument 300 can be utilized. With regard to another embodiment of alight beam assembly, reference is made to FIGS. 21 and 22. Thisembodiment incorporates a “multi-bounce” technique by which the sensingunit 100 being tested is subject to a number of reflected light passes,instead of only one. If an extremely small light beam were used as partof the multi-bounce operation, it could be concluded that the light beamwould reflect from a different spot on the sensing unit 100 on each passof the light. However, the light beam commonly has some finite size, andthe area occupied by the analyte of interest, when present, is typicallyvery small so that it is reasonably concluded that the light beamreflects from a small area on the sensing unit and successivereflections will have some overlap with one or more previous small areasthat were contacted by the light beam. Such a process of overlappingreflections assists in “averaging out” the total surface area of thesensing unit.

[0114] In utilizing the “multi-bounce” technique, the main objective isto produce a sufficient signal above the background noise or othersignals to be detected. One bounce or reflection from the sensing unit100 may not be sufficient to produce such a signal. In order to detect avery thin change in thickness or small change in mass associated withthe sensing unit 100 when the analyte of interest is present, a signalproduced by one bounce or reflection may not be enough. By causing morereflections on passes of light through the sensing unit and adding eachsmall change of polarization to the previous changes (additive orcumulative polarization per reflection on the sensing Unit with analyteof interest when present), then eventually the change in polarizationwill produce a sufficient intensity change, as represented by theresulting signal, so as to be detected. The multi-bounce embodimentdiffers from known prior art in generating multiple reflected signalsthat are at angles other than zero degrees, preferably also differentfrom 90°, to the sensing unit and collecting at least portions of suchreflected light signals and analyzing the total collected light signalin determining whether an analyte of interest is present.

[0115] With reference to FIG. 21, an embodiment of a light beam assembly700 is schematically illustrated that generates multiple reflections anduses partial light transmissions to analyze the intensities of thecollection of such partial transmissions. More specifically, the lightbeam assembly 700 includes a polarized light source 704 that may bemonochromatic or some other acceptable source. A curved mirror 708 isproperly disposed in the path of the light beam from the source 704. Themirror 708 has optical power and special reflective coatings. A quarterwave retarder or compensator plate 712 is positioned in the path of thelight beam that exits the curved mirror 708. The quarter wave retarderplate 712 has an anti-reflective coating on both of its sides. The plate712 is disposed between the curved mirror 708 and a sensing unit 100.The function of the quarter wave retarder plate 712 is to cause thepolarization state that passes through it to be additive afterreflection, and not cause a cancellation of the polarization change, aswill be explained later herein. The light beam assembly 700 alsoincludes a focusing lens/analyzer plate 716 that is in the path of thepartially transmitted light from the curved mirror 708. The focusinglens/analyzer plate 716 can be rotated about its axis in connection withcontrolling the receipt of the transmitted light from the curved mirror708. The focusing lens/analyzer plate 716 is suitably positioned todirect the partially transmitted light from it to a detector unit 720,which collects all of the partially transmitted light for subsequentconversion to a light signal representative of the intensities of thecollected light. This light signal is analyzed, similar to theembodiment previously described, in connection with determining anychange in mass or thickness of the sensing unit 100 due to the presenceof an analyte of interest thereon.

[0116] With respect to a discussion of the number of “bounces” orreflections, as FIG. 21 illustrates, the polarization light source 704directs the light beam to the curved mirror 708. Light from the source704 may or may not pass through the curved mirror 708 and is incidentupon the sensing unit 100 at a first point or spot. Light is reflectedtherefrom and contacts the curved mirror 708 whereby there is a partialtransmission of light A together with a further reflection from thecurved mirror 708 to a different point on the sensing unit 100. Thisresults in a reflection back to the curved mirror 708, where there is apartial transmission of light B, together with another reflection backto the sensing unit 100. Yet another reflection occurs from the sensingunit 100 at a different point thereon to the curved mirror 708, wherepartially transmitted light C passes through the curved mirror 708,while additionally reflected light from the curved mirror 708 isdirected back to the sensing unit 100 at a different point. At thisfurther different point, another reflection occurs from the sensing unit100 to the curved mirror 708 and yet another partial transmission lightD occurs.

[0117] For each of the reflections between the curved mirror 708 and thesensing unit 100, such light passes through the quarter wave retarderplate 712. Each time there is a pass through the plate 712, thepolarization state of the light is 90° plus a small change that occursdue to the thickness of the sensing unit 100. The curved mirror 708, onthe other hand, has special optical properties regarding polarizationand will not introduce any additional polarization and is termed aneutral polarization reflector. Consequently, the only polarizationchange due to the curved mirror 708 is the 180° change due to thereflection. On the other hand, the quarter wave retarder plate 712causes the polarization vector to point in the opposite direction fromthe direction it had when it entered the quarter wave retarder plate712. After two passes through the retarder plate 712 and one reflection,there is a 360° rotation in the polarization state, together with asmall change due to the thickness of the sensing unit 100. In theabsence of the retarder plate 712, the vector sum of the polarizationsat the sensing unit would be 180° out of phase and would cause acancellation of the phase change gained on the reflection at the sensingunit. However, by including the one quarter wave retarder plate 712 tothe optical path, it produces two 90° polarization changes, each timelight passes through first one side thereof and then through the secondside thereof. This summing of polarization changes continues for each ofthe reflections.

[0118] Regarding the generation of the partially transmitted light andthe multiple reflections of the entering light, this is achieved by theoptical power or curvature of the curved mirror surface 708 and theentering angle of incidence of the light beam from the source 704. Thereflective properties of the curved mirror 708 also allow for thepartial transmission of light through the curved mirror 708 each timereflected light strikes the curved mirror 708.

[0119] A further description of this embodiment is provided withreference to the flow diagram of FIG. 22 that is directed to major stepsrelated to the obtaining and analyzing of data related to lightintensities for determining mass changes. In particular, step 750 ofFIG. 22 involves the development of standardized data using a number ofstandard masses and measuring of light intensities, similar to the stepof 550 of FIG. 14 in the previously described embodiment. In accordancewith step 754, the focusing lens/analyzer plate 716 must be properlyoriented for collection of the numerous partial light transmissionsthrough the curved mirror 708. In that regard, the necessary orientationis previously determined and based on the anticipated masses of thesensing units 100. That is, in connection with testing for a specificanalyte of interest, the expected mass range for this analyte ofinterest was previously determined, and the focusing lens/analyzer 716is oriented at an angle based on this previous determination. At step758, power is applied to the light beam source 704, which directs thelight beam to the curved mirror 708. A number of reflections aregenerated, in accordance with step 762 using the sensing unit 100, thequarter wave retarder plate 712 and the curved mirror 708 to therebyproduce a larger polarization that is more readily detectable. In thatregard, at step 766, a sufficient number of partial light transmissionsis generated by which some light from the reflected light on the curvedmirror 708 passes therethrough. At step 770, such partial lighttransmissions are collected using the focusing lens/analyzer plate 716.The numerous partial light transmissions are then detected by thedetector unit 720 at step 774. The detector unit 720 uses theintensities of the polarized light to generate a light signal thatrelates to the mass of the sensing unit 100. At step 778, the value ofthis sum light signal is compared with the previously determinedstandard data as part of determining whether or not the analyte ofinterest is present.

Test Piece Containing Device

[0120] In one embodiment, with reference to FIGS. 23 and 24, the systemalso includes device 800 for containing a test piece having a number ofsensing units 100 to be tested. Unlike known prior art, the device 800includes a combination of assemblies and elements that are used, eitheralone or together, to precisely control heating, humidity,cross-contamination and mixing of materials with or part of sensingunits 100. The device 800 includes an enclosure unit 804 (FIG. 15) and abase plate 808 for housing a number of assemblies useful in providingsuch functions. An insulating cover 806 is preferably provided over theenclosure unit 804 in order to reduce the amount of heat loss to theenvironment outside of the device 800, which effectively reduces theconvection inside of the device 800. When convection is reduced, thereis less unwanted evaporation. An inner lid 810 is also provided with theinsulating cover 806. The inner lid 810 geometrically influences theconvection pattern created by a temperature gradient caused by theheating assembly 812 (higher temperature) and the walls (lowertemperature) of the device 800. The inner lid 810 is curved in way thatprevents condensation from dripping down onto the sensing units and testpiece. The base plate 808 has a sealing ring 814 (FIG. 24) locatedaround its edge which forms a seal between the base plate 808 and theinsulating cover 806. This reduces the amount of heat and humidity lossfrom the device 800 where the base plate 808 and the cover 806 come intocontact.

[0121] The heating assembly 812 is used to heat the sensing units on thetest piece 332 to a desired or predetermined temperature, such as 37degrees Celsius and maintain that temperature to within at least ±1degree Celsius. The heating assembly 812 includes a plate sub-assembly816 that is comprised of a heating element situated between twostainless steel plates. These two plates act as heat reservoirs and areessentially in direct contact with the test piece and, by thisarrangement, the heating assembly 812 provides heat to the sensing unitsby conduction. In order to generate the heat, the plate sub-assembly 816is powered by a heater controller (not shown) that is electricallyconnected to the plate sub-assembly 816 by a power cord 818 (FIG. 23).The heating assembly 812 also includes a support frame 820 thatsurrounds at least some of the periphery of the plate sub-assembly 816.The support frame 820 is connected to lower hinge members 824 a, 824 bat one end of the support frame 820. The lower hinge members 824 a, 824b enable the heating assembly 812 to be pivoted for desired access tothe space below the heating assembly 812, as will be understood from asubsequent description of the humidifier assembly of the device 800.Disposed upwardly of the heating assembly 812 is a barrier manifold 830that is an elongated member having a number of barrier slits 834 thatare perpendicular to the elongated plane of the barrier manifold 830.The barrier slits 834 have a sufficient depth to receive barrier members838 so that portions of the barrier members 838 are held in the barrierslits 834 and other portions of the barrier members 838 extend outwardlyfrom the plane of the barrier member 830. One such barrier member 838 isshown in FIG. 23. The barrier members 838 are spaced a predetermineddistance from each other and such spacing is based on the spacingbetween sensing units on a test piece. That is, the barrier manifold 830overlies the test piece and each of the sensing units is separated fromeach of the other sensing units by one or more barrier members 838. Inone embodiment, the barrier members 838 include O-rings which contactthe body of the test piece without contacting the area where the test isto be taken. Hence, the barrier manifold 830 with barrier members 838prevents cross-contamination of the sensing units 100. The barriermanifold 830 also allows the sensing units to the washed and dried whilein the device 800. The barrier manifold 830 is connected at its ends toupper hinge members 842 a, 842 b. At their opposite end portions, theupper hinge members 842 a, 842 b are connected to manifold arms 850 a,850 b. This arrangement enables the barrier manifold 830 to be pivotedrelative to the plate sub-assembly 816. When a test piece is to beplaced in the device 800, the barrier manifold 830 is pivoted away fromthe plate sub-assembly 816 and the test piece is able to be placed onthe plate sub-assembly 816. Then, the barrier manifold 830 is pivoteddownwardly over the test piece 332 and provides a slight clampingpressure using the barrier members 838 in order to safeguard againstcross-contamination.

[0122] The device 800 further includes a humidifier assembly 860 that iscomprised of a number of absorbative members for maintaining a relativehumidity of 100% within the enclosure unit 804 and the base plate 808 inorder to avoid unwanted loss of sensing unit materials due toevaporation. The absorbative members include a main absorbative member864 that is positioned in a cavity 868 of a base member 872 that issupported on the base plate 808. The main absorbative member 864 has alength that extends for at least a substantial portion of the length ofthe plate subassembly 816 and is positioned essentially directly belowthis sub-assembly. The main absorbative member 864 is soaked with waterprior to its placement in the cavity 868. Two side absorbative members876 a, 876 b are also utilized and are located to the side of the mainabsorbative member 864 adjacent to and on top of a number of wells 880formed in a wall 884 of the base member 872. The side sponge members 876a, 876 b are also soaked with water before placing them in the device800 next to the wells 880. During use, the side sponge members 876 a,876 b draw water from the wells 880 by means of a wicking operation tomaintain their soaked or moisture-laden state in order to continue toprovide the desired humidity.

[0123] The device 800 also includes a mixing assembly 892(diagrammatically illustrated in FIGS. 23 and 24) which is provided toenhance the mixing of reagents associated with the sensing units 100 tobe analyzed. In that regard, and with reference to a single drop ofmaterial(s) on the sensing unit 100, the motion and pattern of diffusionhas been observed to be a slow descending spiral that varies with thelocation and orientation of the mixing assembly 892. When this motionwithin a drop is added to the motion provided by convection due to thedrop being heated, there is greater fluid movement and mixing withineach drop. In the preferred embodiment, the mixing assembly 892 includesa mechanism for providing oscillatory motion using an offset weightrotating about a fixed axis. This rotation is accomplished by aminiature variable-speed motor. Significantly, as illustrated in FIG.23, the mixing assembly 892, including the motor and the offset weight,is situated in a compound axis orientation next to the base member 872along its length. Preferably, the orientation angle is greater thanabout 10 degrees but less than about 70 degrees relative to the plane ofthe base plate 808.

[0124] In view of the foregoing description, a system is described thatincludes a number of sub-system components that cooperate with eachother in the detection and/or measurement of an analyte of interestusing mass change. The system components include a test piece on which anumber of sensing units are located that are to be tested in connectionwith determining whether one or more of them has an analyte of interest.The test piece is dimensioned to be held in a test piece containingdevice that provides a number of functions in the preparation and careof the test piece with sensing units for subsequent testing. Suchfunctions include precisely controlling the heating, humidifying,avoidance of cross-contamination and mixing of materials that are withor part of the sensing units. The sensing units include an attachmentlayer that is able to resist delamination of the different layers. Thesensing unit may include an attachment layer that is used to immobilizea ligand layer that is receptive to the analyte of interest. The sensingunit may include a tripartite and/or dual element attachment layer thatis characterized by an insulating layer located between an upper surfaceand a lower surface. The insulating layer acts to prevent the transferof unwanted effects between the lower and upper binding surfaces. Thesensing unit may include the dual element attachment layer in which thelower element has an organofunctional silial compound. Mass enhancementsystems are preferably utilized that can include a variety of materialssuch as kinetic-active mass enhancement with one or more desired enzymesassociated with the substrate, passive mass enhancement systems in whichan existing mass/constant is used and/or a self-assembling amplificationsystem. A further system component is the instrument that is utilized indetermining whether or not the analyte of interest is present on aparticular sensing unit. The instrument is highly sensitive and able toaccurately determine whether an analyte is present. Such an instrumentmay include components for generating and analyzing multiple reflectionsfrom the same sensing unit to enhance or amplify the detected signal.The instrument may include optical elements by means of which polarizedlight having only one component is generated to enhance the detectedsignal that is indicative of the presence of the analyte of interest.

[0125] The foregoing discussion of the invention has been presented forpurposes of illustration and description. Further, the description isnot intended to limit the invention to the form disclosed herein.Consequently, variations and modifications commensurate with the aboveteachings, within the skill and knowledge of the relevant art, arewithin the scope of the present invention. The embodiments discussedhereinabove are further intended to explain the best mode known ofpracticing the inventions and to enable others skilled in the art toutilize the inventions in such, or in other embodiments and with thevarious modifications required by their particular application or usesof the inventions. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

What is claimed is:
 1. A sensing unit involved in detecting an analyteof interest, comprising: a substrate having an outer surface; anattachment layer for adhering to said substrate, said attachment layerused for controlled binding of at least a first material and in whichsaid substrate outer surface has properties that cause unwanted effectson the first material, said attachment layer including: a lower bindingsurface having a material composition that adheres to said substrate inorder to provide a durable and stable lamination of said attachmentlayer to said substrate; an upper binding surface having a materialcomposition different from said material composition of said lowerbinding surface, said upper binding surface including binding propertiesassociated with said first material which requires a controlledimmobilization environment to retain its functional characteristics; andan insulating layer disposed between said lower binding surface and saidupper binding surface, said insulating layer having a materialcomposition different from each of said material compositions of saidlower binding surface and said upper binding surface and in which saidinsulating layer acts to prevent the transfer of unwanted effects fromsaid lower binding surface to said upper binding surface and from saidupper binding surface to said lower binding surface; wherein each ofsaid material compositions of said lower binding surface and said upperbinding surface has properties that are enhanced by the use of saidinsulating layer.
 2. A sensing unit, as claimed in claim 1 , furtherincluding: at least one capture layer joined to said attachment layerfor providing an environment in which the analyte of interest, whenpresent, is properly immobilized to permit its subsequent detection,said capture layer including said first material.
 3. A sensing unit, asclaimed in claim 1 , wherein: each of said material compositions of saidlower binding surface and said upper binding prevent it from being usedfor said material composition of said insulating layer.
 4. A sensingunit, as claimed in claim 1 , wherein: said substrate is other than afree electron metal.
 5. A sensing unit, as claimed in claim 1 , wherein:one of said lower binding surface and said upper binding surfaceconsists essentially of inorganic monovalent compounds having linearbackbone structures.
 6. A sensing unit, as claimed in claim 1 , wherein:one of said lower binding surface and said upper binding surfaceconsists essentially of at least one of organic monovalent andpolyvalent compounds and in which said compounds have particles thatretain their particle size of at least 40 microns or do not have saidparticles.
 7. A sensing unit, as claimed in claim 1 , wherein: saidlower binding surface consists essentially of an organofunctional silialcompound.
 8. A sensing unit, as claimed in claim 1 , wherein: saidattachment layer includes some of the following materials:6-azidosulfonylhexytriethoxy silane, aminoethyl-aminopropyl trimethoxysilane, aminopropyltrioxysilane, 3-isocyanatopropyltriethoxysilane,phenyltriethoxysilane, poly(ethylene)glycol, poly(ethylene)oxide,nitrocellulose, paralene, nylon, polyester, polyamides, polyurethane,polystyrene, avidin (and its derivatives), and biotin.
 9. A sensingunit, as claimed in claim 1 , wherein: said insulating layer consistsessentially of one or more materials from the following group:polystyrene, polyurethane, and polyethylene glycol.
 10. A sensing unit,as claimed in claim 7 , wherein: said organofunctional silial compoundincludes amino-silane, azido-silane and silanes comparable thereto. 11.A sensing unit, as claimed in claim 1 , wherein: said substrate consistsessentially of a crystalline silicon.
 12. A sensing unit, as claimed inclaim 1 , wherein: said first material i s a specific material.
 13. Asensing unit, as claimed in claim 1 , further including: massenhancement means for amplifying a signal for detection related to thepresence of the analyte of interest.
 14. A sensing unit, as claimed inclaim 12 , wherein: said mass enhancement means includes one or moreenzymes associated with such attachment layer.
 15. A sensing unitinvolved in detecting an analyte of interest, comprising: a substrateother than a free electron metal and having an outer surface; anattachment layer for adhering to said substrate, said attachment layerused for controlled binding of at least a first specific material and inwhich said substrate outer surface has properties that cause unwantedeffects on the first specific material, said attachment layer including:a lower element having a material that adheres to said substrate inorder to provide a durable and stable lamination of said attachmentlayer to said substrate, said lower element consisting essentially of anorganofunctional silial compound; and an upper element having a materialcomposition different from said material composition of said lowerelement, said upper element including an outer surface including bindingproperties associated with said first specific material which requires acontrolled immobilization environment to retain its functionalcharacteristics; at least one capture layer joined to said attachmentlayer for providing an environment in which the analyte of interest,when present, is properly immobilized to permit its subsequentdetection, said capture layer including said first specific material;wherein said material composition of said upper element has propertiesthat prevent it from being used for said material composition of saidlower element.
 16. A biosensor device, as claimed in claim 15 , wherein:said organofunctional silial compound includes amino-silane,azido-silane and silanes comparable thereto.
 17. A sensing unit, asclaimed in claim 15 , wherein: said upper element consists essentiallyof one or more of the following: polystyrene, polyurethane andpolyethylene glycol.
 18. A sensing unit, as claimed in claim 15 ,further including: mass enhancement means associated with saidattachment layer for amplifying detection of the analyte of interest.19. A sensing unit, as claimed in claim 18 , wherein: said massenhancement means includes one or more enzymes provided with said upperelement.
 20. A sensing system for obtaining information related tosensing units, comprising: a test piece having a number of sensing unitsand indicating means associated with each of the sensing units; ahousing having a number of walls and a receiver slot for receiving saidtest piece wherein said test piece can move along a path of saidhousing; a light beam assembly for obtaining information from eachsensing unit for use in determining the presence of a specific substanceof interest; and controller means located within said housing forcausing said test piece to move relative to said housing path.
 21. Asystem, as claimed in claim 20 , wherein: said indicating means includesindicia and said controller means includes a reader assembly for readingsaid indicia in connection with stopping movement of said test piecealong said housing path.
 22. A system, as claimed in claim 21 , wherein:said indicia includes an identification code that identifies at leastone of the sensing units.
 23. A system, as claimed in claim 20 ,wherein: said controller means comprises a test piece control assemblythat includes a motor disposed within said walls of said housing.
 24. Asystem, as claimed in claim 20 , further including: an input unitprovided on one of said walls of said housing for inputting informationrelated to the sensing units.
 25. A system, as claimed in claim 20 ,further including: display means located on one of said walls of saidhousing for displaying information associated with the sensing units.26. A system, as claimed in claim 20 , further including: data memoryfor storing data related to each of the sensing units includingidentification data, time-related data and data related to whether ornot the analyte of interest is present.
 27. A system, as claimed inclaim 20 , further including: at least a first port communicating withan external computer system by which said external computer system candownload software for execution by a processor of said controller means.28. A system, as claimed in claim 20 , wherein: said controller meansincludes a processor for determining values related to the sensing unitsand whether or not an analyte of interest is present for each of thesensing units.
 29. A system, as claimed in claim 20 , wherein: saidindicating means includes at least one of a mark to indicate a locationof each of the sensing units and a code for identifying at least one ofthe sensing units.
 30. A system, as claimed in claim 20 , wherein: saidcontroller means includes a number of electronic components including aprocessor and in which said electronic components are held on a printedcircuit board and in which said printed circuit board is located betweensaid light beam assembly and an input unit for providing inputinformation used by said processor.
 31. A system, as claimed in claim 20, wherein: said light beam assembly includes polarizer means forreceiving polarized light having only one component when each sensingunit is tested to determine whether the sensing unit has the analyte ofinterest and in which said polarizer means remains stationary when saidpolarized light is being received.
 32. A system, as claimed in claim 20, wherein: said light beam assembly generates a number of reflectedlight beams having intensities for each of the sensing units, with eachof said reflected light beams being produced at an angle different fromperpendicular to each of the sensing units.
 33. A system, as claimed inclaim 32 , wherein: said light beam assembly includes a curved mirrorthat reflects and transmits light and wave retarder means for providingadditive polarization of said reflected light intensities for each ofthe sensing units to enhance sensitivity for determining the presence ofthe analyte of interest.
 34. A method for obtaining assay data,comprising: providing a test piece having a number of sensing units;controlling movement of said test piece; checking for identifying meanson said test piece; discontinuing movement of said test piece based onsaid checking step; obtaining data related to a first sensing unit onsaid test piece including determining whether an analyte of interest ispresent using an optical detecting apparatus; and storing informationrelated to said determining step including identification data relatedto the first sensing unit, time-related data, and a determined valuerelated to whether the analyte of interest is present with the firstsensing unit.
 35. A method, as claimed in claim 34 , wherein: saidcontrolling step includes regulating a motor using a processor thatreceives a signal based on said identifying means.
 36. A method, asclaimed in claim 34 , wherein: said identifying means includes at leastone of a first code that identifies a first sensing unit and alignmentindicia that indicates a position of at least one of the sensing units.37. A method, as claimed in claim 34 , wherein: said providing stepincludes inserting said test piece into a receiver slot of a housingthat contains said optical detecting apparatus and, before saidinserting step, locating said indicating means on said test piece.
 38. Amethod, as claimed in claim 34 , wherein: said controlling step includesregulating the turning on and turning off of a motor using a processorand contacting said test piece using means for engaging driven by saidmotor in order to move said test piece.
 39. A method, as claimed inclaim 34 , further including: inputting identification informationrelated to at least one of the sensing units using an input unitprovided on one of the walls of a housing that contains said opticaldetecting apparatus.
 40. A method, as claimed in claim 34 , wherein:said obtaining step includes obtaining data for all of the sensing unitson said test piece and maintaining an angular position of polarizermeans of said optical detecting apparatus during receiving of lightintensities for all of the sensing units on said test piece, with saidlight intensities being used to provide said data for all of the sensingunits.
 41. A method, as claimed in claim 34 , wherein: said obtainingstep includes collecting at least portions of light related to a numberof reflected light beams having intensities that are reflected fromareas of the first sensing unit and in which each incident light beamfrom which each of said reflected light beams are generated is at anangle other than perpendicular to the first sensing unit.
 42. A devicefor heating and providing humidity to a number of sensing unitscontained on a test piece, comprising: a heating assembly for heatingsensing units when a test piece containing them is provided with saiddevice; a humidifier assembly communicating moisture to said heatingassembly for humidifying the sensing units; a barrier manifold forsubstantially preventing cross-contamination of the sensing units whenthe test piece containing them is provided with said device; and amixing assembly for mixing of materials in the sensing units when thetest piece containing them is provided with said device.
 43. A device,as claimed in claim 42 , wherein: said heating assembly includes a platesub-assembly that is heated for providing heat by conduction to the testpiece when it is provided with said device.
 44. A device, as claimed inclaim 42 , wherein: said heating assembly includes a support frame thatis joined to said plate sub-assembly with both of said support frame andsaid plate sub-assembly being pivotal.
 45. A device, as claimed in claim42 , wherein: said humidifying assembly includes water holding meanslocated below said heating means.
 46. A device, as claimed in claim 45 ,wherein: said water holding means includes at least a first spongemember positioned to a side of said heating assembly and at least onewell for containing water adjacent to said first sponge member.
 47. Adevice, as claimed in claim 42 , wherein: said barrier manifold includesa number of spaced barrier members that act to separate each of thesensing units from the other sensing units when the test piececontaining the sensing units is positioned in said device.
 48. A device,as claimed in claim 42 , wherein: said barrier manifold is pivotal andapplies a pressure to the test piece when the test piece is positionedin said device.
 49. A device, as claimed in claim 42 , wherein: saidmixing assembly includes a motor that is positioned at an angle toenhance the mixing of the materials in the sensing units.
 50. A device,as claimed in claim 49 , wherein: said angle is greater than about 10degrees but less than about 70 degrees.
 51. An instrument for detectingan analyte of interest, comprising: first means for supporting a sensingunit to be tested; second means for locating the sensing unit in adesired position; and third means for determining whether the analyte ofinterest is present with the sensing unit being tested, said third meansincluding an optical detecting apparatus for generating a number oflight reflections from the sensing unit, with each of said lightreflections being at an angle other than 0°, 90° and Brewster's anglerelative to said first means, said third means including collectionmeans for collecting at least portions of said light reflections andusing said collected light reflections in determining whether theanalyte of interest is present.
 52. An instrument, as claimed in claim51 , wherein: said means for generating includes a mirror and a waveretarder plate, said wave retarder plate being disposed in a path ofsaid light reflections between the sensing unit and said mirror andwherein said wave retarder plate causes said light reflections that passthrough it to be additive of polarization change.
 53. An instrument, asclaimed in claim 52 , wherein: said mirror includes curved portions andin which said mirror has optical power and a reflective coating, withportions of said light reflections received by said mirror beingtransmitted through said mirror.
 54. An instrument, as claimed in claim52 , wherein: said mirror has a reflective coating and said waveretarder plate is a portion of said reflective coating.
 55. Aninstrument for detecting an analyte of interest, comprising: first meansfor supporting a sensing unit to be tested; second means for locatingthe sensing unit in a desired position; and third means for determiningwhether the analyte of interest is present with the sensing unit, saidthird means including an optical detecting apparatus that has means forgenerating polarized light having only one component and means forextinguishing said one component, wherein said means for generating andsaid means for extinguishing remain fixed in position during the timethat said optical detecting apparatus provides light to the sensingunit.
 56. An instrument, as claimed in claim 55 , wherein: said onecomponent includes one of s-polarization and p-polarization.
 57. Aninstrument, as claimed in claim 55 , wherein: said means for generatingincludes a linear polarizer that receives light prior to said lightbeing incident upon the sensing unit and said means for extinguishingincludes a detector polarizer that receives light reflected from thesensing unit.